Synthesis, analysis of molecular and crystal structures, estimation of intermolecular interactions and biological properties of 1-benzyl-6-fluoro-3-[5-(4-methylcyclohexyl)-1,2,4-oxadiazol-3-yl]-7-(piperidin-1-yl)quinolin-4-one

The synthesis of the potential antimicrobial and antiviral drug, 5-[1-benzyl-6-fluoro-7-(piperidin-1-yl)-quinolin-4(1H)-on-3-yl]-3-(4-methylcyclohex-1-yl)-1,2,4-oxadiazole, was proposed. Its molecular and crystal structures were defined and described, whereas the biological activity was predicted with molecular docking.

The title compound, C 30 H 33 N 4 O 2 F, can be obtained via a two-step synthetic scheme involving 1-benzyl-6-fluoro-4-oxo-7-(piperidin-1-yl)-1,4-dihydroquinoline-3-carbonitrile as a starting compound that undergoes substitution with hydroxylamine and subsequent cyclization with 4-methylcyclohexane-1-carboxylic acid. It crystallizes from 2-propanol in the triclinic space group P1 with a molecule of the title compound and one of 2-propanol in the asymmetric unit. After the molecular structure was clarified using NMR and LC/MS, the molecular and crystalline arrangements were defined with SC-XRD. A Hirshfeld surface analysis was performed for a better understanding of the intermolecular interactions. One strong (O-HÁ Á ÁO) and three weak [C-HÁ Á ÁF (intramolecular) and two C-HÁ Á ÁO] hydrogen bonds were found. The contributions of short contacts to the Hirshfeld surface were estimated using two-dimensional fingerprint plots showing that OÁ Á ÁH/HÁ Á ÁO, CÁ Á ÁH/HÁ Á ÁC and CÁ Á ÁC contacts are the most significant for the title compound and OÁ Á ÁH for the 2-propanol. The crystal structure appears to have isotropically packed tetramers containing two molecules of the title compound and two molecules of 2-propanol as the building unit according to analysis of the distribution of pairwise interaction energies. A molecular docking study was carried out to evaluate the interactions of the title compound with the active centers of macromolecules corresponding to viral targets, namely, anti-hepatitis B activity [HBV, capsid Y132A mutant (VCID 8772) PDB ID: 5E0I] and anti-COVID-19 main protease activity (PDB ID: 6LU7). The data obtained revealed a noticeable affinity towards them that exceeded that of the reference ligands.

Chemical context
One of the promising areas of investigation in the search for new antibiotic compounds is the synthesis of fluoroquinolone derivatives. These purely synthetic compounds have been known since 1962 when the first drug from this group, nalidixic acid, was discovered. The scope of their application has changed and substantially broadened from that time since fluorine atoms were included in the 6-position of the quinoline molecule. Today, fluoroquinolones have positive pharmacokinetic properties, high oral bioavailability, a wide spectrum of action, and good tolerability (Ezelarab et al., 2018). At present, four generations of fluoroquinolones exist , the last of which has found successful use even in the treatment of bacterial pneumonia that developed against the background of COVID-19 (Beović et al., 2020). Fluoroquinolones also show their own antiviral potential (Xu et al., 2019;Cardoso-Ortiz, et al., 2023), which opens up prospects for their use in mixed infections. Hence today there is interest in new fluoroquinolones as potential simultaneous antibacterial and antiviral agents.
One of the ways to create fluoroquinolones with combined action is the synthesis of hybrid structures that contain several pharmacophores. The basic fluoroquinolone molecule can be structurally modified in several directions at once, which significantly expands both the synthetic possibilities and the opportunities for further research into the biological activity of new compounds (Suaifan & Mohammed, 2019). Structural modification of the bicyclic system of fluoroquinolones is possible due to the substitution of a nitrogen atom in position 1, a carboxyl group in position 3, an oxo group in position 4, a fluorine atom in position 6 and by hybridization with heterocyclic nitrogen fragments in position 7 (see scheme). Thus, hybrids of fluoroquinolones with derivatives of phenylthiazole, quinazoline, thiazolidine, thiadiazole, pyrimidine, dithienylethene, and 1,2,4-triazole are widely described in the literature (Suaifan & Mohammed, 2019;Jia & Zhao, 2021). Our scientific team has been fruitfully working in this direction (Bylov et al., 1999;Silin et al., 2004;Savchenko et al., 2007;Hryhoriv et al., 2022;Vaksler et al., 2022). At the same time, a thorough analysis of literature sources demonstrates that modification of the 3-position of fluoroquinolones is promising and insufficiently researched (Oniga et al., 2018). We therefore decided to broaden our investigations of these compounds with studies of 5-[1-benzyl-6-fluoro-7-(piperidin-1-yl)-quinolin-4(1H)-on-3-yl]-3-(4-methylcyclohex-1-yl)-1,2,4-oxadiazole.

Supramolecular features
Regarding the van der Waals radii proposed in Bondi, 1964 for all the atoms except hydrogen (Rowland & Taylor, 1996), a very weak non-classical intramolecular hydrogen bond C12-H12AÁ Á ÁF1 involving the carbon atom from the piperidine moiety (Table 1) is found together with the three inter-molecular hydrogen bonds of two types. The first type is the strong bond O-HÁ Á ÁO between the hydroxylic group of isopropyl alcohol and the keto oxygen atom of the main molecule. The isopropyl alcohol molecule is disordered over two positions (A and B) in a 0.655 (8):0.345 (8) ratio due to a rotation around this hydrogen bond. The weak non-classical C-HÁ Á ÁO hydrogen bonds involving the oxygen atom of the isopropyl alcohol and methylene groups from the piperidine ring and benzyl moiety belong to the second type. While the bond C16-H16BÁ Á ÁO1A exists only for the disordered position A, the bond involving the benzyl moiety exists for both disordered positions of the solvent molecule: C17-H17BÁ Á ÁO1A and C17-H17BÁ Á ÁO1B. In addition,stacking between the quinoline fragments of the original molecule and its symmetry equivalent at Àx, Ày + 1, Àz + 2 [regarding the short contacts C3Á Á ÁC6 = 3.379 (4) Å and C5Á Á ÁC10 = 3.392 (4) Å ] should be mentioned. The molecules of the title compound are bound in pairs withstacking (as the pair of symmetry equivalents x, y, z and Àx, Ày + 1, Àz + 2) and the pairwise interactions are complemented by the strong O-HÁ Á ÁO and weak C-HÁ Á ÁO hydrogen bonds involving two isopropyl alcohol molecules (symmetry operations: x À 1, y + 1, z and Àx + 1, Ày, Àz + 2). Therefore, the strongly bound tetramers (Fig. 2) consisting of two molecules of the title compound and two molecules of isopropyl alcohol exist in the crystal despite the fact of disorder.

Hirshfeld surface analysis
A Hirshfeld surface analysis (Spackman & Byrom, 1997) was applied to the studied structure. Originally, it was a method that allows the crystal space to be distributed into the regions belonging to different molecules, i.e. regions where the specified promolecular electron density exceeds the procrystal density. It was modernized with 2D-fingerprint plots in Crys-talExplorer17 (Spackman et al., 2021) and can be used for the evaluation of intermolecular interactions. The standard 'high' resolution was applied in this study. Four regions with d norm significantly lower than the van der Waals contact length (in red) emerge on the surface of the title compound ( Fig. 4a) and two more regions exhibit for the isopropyl alcohol (Fig. 4b).

Figure 2
Tetrameric building unit bonding: hydrogen bonds (in cyan) and short contacts (in magenta).

Figure 3
Comparison of hydrogen bonds (in cyan) and short contacts (in magenta) of the 2-propanol molecules in positions A and B. intermolecular contacts described above is visible (Fig. 4c,d).
As expected, the Hirshfeld surface of the isopropanol repeats the contacts O-HÁ Á ÁO and C16-H16BÁ Á ÁO1A. Thereby, the appearance of the tetramers described above is also confirmed using Hirshfeld surface analysis, especially after renormalization. The 2D-fingerprint plots showed five types of intermolecular contacts whose contribution into the Hirshfeld surface area for the title compound exceeds 5.0%. They are HÁ Á ÁH, 61.9%; CÁ Á ÁH, 11.3%; OÁ Á ÁH, 9.4%; NÁ Á ÁH, 5.3%, and FÁ Á ÁH, 5.0%. However, just three of them relate to areas with the values of the internal and external distances (d i and d e ) below the van der Waals radii of the corresponding atoms ( Fig. 5a-c). Sharp peaks, whose appearance is usually associated with the formation of intermolecular interactions, are found for the CÁ Á ÁH and OÁ Á ÁH contacts, as well as for the 'less significant' CÁ Á ÁC contact (3.9% of the area). They point out the hydrogen bonds and stacking interactions in a manner similar to the conventional supramolecular analysis. Similarly to it, the three contributions exceeding 5.0% of the Hirshfeld surface area are found for isopropyl alcohol: HÁ Á ÁH, 78.1%; OÁ Á ÁH, 15.3%, and NÁ Á ÁH, 5.7%; with just one type below the van der Waals radii of the corresponding atoms: OÁ Á ÁH (Fig. 5d). The contributions of intermolecular contacts do not differ significantly for positions A and B, except for the appearance of the short contact C2BÁ Á ÁC2B 0 (Fig. 6) on the Hirshfeld surface.

Analysis of the pairwise interaction energies
The topological analysis allowed us to construct a model of the intermolecular interactions in a crystal. However, this model cannot be confirmed without an assessment of the energetic structure and the contributions of various interactions: hydrogen bonds, as obviously strong classical ones, as nonclassical ones with a variable and often underestimated strength (Sutor, 1962;Desiraju, 1996Desiraju, , 2005, continuously underrated stacking (Dharmarwardana et al., 2021;Shishkina et al., 2019;Zhao & Truhlar, 2008) and non-specific interactions. The procedure proposed by Konovalova et al. (2010) and Shishkin et al. (2012) was applied to define the pairwise interaction energies of the molecules in crystals in a two-step procedure considering the molecule and the stacking-dimer of molecules as a building unit on a par with the molecule of isopropyl alcohol. The pairs were formed containing the central building unit and its neighboring building units from the first coordination shell. Calculations of the interaction energies for each pair were performed using the B97 functional (Becke, 1997;Schmider & Becke, 1998) with the parameterized three-body (D3) dispersion correction (Grimme et al., 2010) and Becke-Johnson dumping (Grimme et al., 2011). The basis set def2-TZVP was used (Weigend & Ahlrichs, 2005; Weigend, 2006) and the basis set superposition error (BSSE) correction was implemented according to the Boys-Bernardi counterpoise scheme (Boys & Bernardi, 1970) in the software package ORCA 3.0.3 (Neese et al., 2020). Energy vector diagrams were used for the visualization of the calculated interaction energies in a standard way (Shishkin et al., 2012(Shishkin et al., , 2014. In addition to this, the interaction energy decomposi-tion was performed using an 'accurate' energy model in the program CrystalExplorer17 for the model with the molecules as building units to clarify the nature of the interactions. The interactions in the stacking-bonded dimer of the title compound turned out to be two times stronger than any other interaction of an individual molecule in the crystal structure ($33.0 kcal mol À1 ). The dispersion is many times superior to [192][193][194][195][196][197][198][199][200] research communications Table 2 Symmetry codes, binding types and interaction energies (kcal mol À1 ) of the building units (BU) (single molecules) with neighbors.

Molecular docking
To estimate the potential biological properties and the possible interactions of the title compound with the active centers of target viral macromolecules, we conducted a molecular docking study. Two targets from the Protein Data Bank (PDB) were utilized for this purpose. The first one is the capsid of the Hepatitis B virus (HBV capsid Y132A mutant VCID 8772, PDB ID: 5E0I; Klumpp, et al., 2015). The second is COVID-19 main protease PDB ID: 6LU7 (Jin et al., 2020). The crystal structure of the HBV capsid Y132A contains 157 amino acid residues; the molecular weight is 109.09 kDa. Methyl 4-(2-bromo-4-fluorophenyl)-6-(morpholin-4-ylmethyl)-2-(1,3-thiazol-2-yl)pyrimidine-5-carboxylate was used as a reference ligand. For 5E0I there are six protein chains designated as A, B, C, D, E, and F. According to our computations, the residual mean squared deviation between experimental data from X-ray diffraction analysis and the docking-generated position is around 1 Å , which is even better than the X-ray resolution reported for the structures (1.95 and 2.16 Å , respectively). Hence, for the docking procedure, we can use any of the above-mentioned chains. We used chain A in the actual calculations.
These target macromolecules had previously been utilized for similar research, and therefore we proceeded with them both to obtain the docking results, and determine whether we could enhance the antiviral properties that were observed for some fluoroquinolones that had been hybridized with heterocycles.
For the graphical analysis, the free software packages Jmol (Jmol, 2022) and PyMol (DeLano, 2002) were used. The virtual screening, pharmacophore investigation, and molecular docking procedures, with the subsequent analysis of their data, were performed using the LigandScout 4.4 software complex (Wolber & Langer, 2005). For the calculations of standard molecular QSAR parameters, the popular resource SwissADME from the Swiss Institute of Bioinformatics (Daina et al., 2017) was utilized.
According to the results of docking studies, it was found that the tested molecule has a significant affinity to both targets (Table 4). This is evidenced by the values of the scoring functions and the free binding energy in comparison to the values of the described reference ligands. Among the QSAR properties obtained from SwissADME, we included only the most important, namely MlogP (calculated by using the Moriguchi approach), LogS (by ESOL) and topological polar surface area (TPSA) (Daina, et al., 2017). These parameters are important characteristics of the transport properties of a drug through membranes. While the LogS and TPSA parameters correspond to the drug likeliness criterion, the lipophilicity for the title compound is noticeably larger. However, this difficulty can potentially be eliminated by some structural chemical modifications, for example, by incorporating appropriate substituents.
An analysis of the geometric location in the active sites of the selected targets showed that the formation of complexes is facilitated by hydrogen bonds (shown with dotted red arrows) and hydrophobic (van der Waals) intermolecular interactions (designated in yellow) (Fig. 8)

Figure 8
Interactions (on the left) and configuration of the title compound (on the right) within the active centers of target viral macromolecules (5E0I -at the top, 6LU7 -at the bottom).
in hydrogen bonds (in Å ) are presented in the left part of this figure. It can be seen that the obtained lengths are quite large, but fall within the typical range for hydrogen bonds (2.5-4.0 Å ). Therefore, the investigated compound is promising for further in vitro research of both the antimicrobial and antiviral activity.

Synthesis and crystallization
The starting reagents are commercially available and, as well as solvents, were purchased from Sigma Aldrich and were used without further purification.
Compound 1 (1 mmol) and N,N 0 -carbonyldiimidazole (1.1 mmol) were dissolved in N,N-dimethylformamide and stirred at 373 K for 20 minutes. After that hydroxylamine (1.1 mmol) was added and heating was maintained for 6 h. The mixture was cooled to room temperature, then water was added, and the obtained precipitate was filtered and recrystallized from an isopropanol-DMF mixture. Then 1 mmol of the obtained compound (2), N,N 0 -carbonyldiimidazole (1.1 mmol) (compound 3) and 4-methylcyclohexane-1-carboxylic acid (1 mmol) (compound 4) were dissolved in N,Ndimethylformamide (80 mL). The mixture was stirred at 333 K for 1 h. The mixture was then cooled to room temperature and the obtained precipitate was filtered, washed with ethanol and recrystallized from an ethanol-DMF mixture.
Further crystallization by slow evaporation of a solution in isopropanol was carried out to provide single block-like colorless crystals suitable for X-ray diffraction analysis (m.p. 476-477 K).

NMR and LC/MS characterization
The NMR spectra were recorded on a Varian MR-400 spectrometer with standard pulse sequences operating at 400 MHz for 1 H NMR and 101 MHz for 13 C NMR. For the NMR spectra, DMSO-d 6 was used as a solvent. Chemical shift values are referenced to residual protons ( 2.49 ppm) and carbons ( 39.6 ppm) of the solvent as an internal standard.

Refinement
Crystal data, data collection and structure refinement details are summarized in Table 5. All hydrogen atoms were refined using a riding model with U iso = nU eq of the carrier atom (n = 1.5 for methyl and hydroxyl groups and n = 1.2 for other hydrogen atoms). During the refinement, the distances between the atoms of the disordered isopropyl alcohol were restrained to the following values: 1.524 Å for bonds C1A-C2A, C1B-C2B, C1A-C3A, C1B-C3B with an estimated standard deviation of 0.015 Å (according to Dunitz & Bü rgi, 1994) and 1.432 Å for bonds O1A-C1A/O1B-C1B with an estimated standard deviation of 0.011 Å . The atoms of each disordered position of the isopropyl alcohol were restrained to have the same U ij components with an estimated standard deviation of 0.01 Å 2 (0.02 Å 2 for terminal atoms). They were subject to a 'rigid bond' restraint as well with an estimated standard deviation of 0.0025 Å 2 .